HCV dependencies on the host machinery are both intricate and extensive. Each of these host dependencies is a potential therapeutic target. Previous efforts have been successful in discovering important steps in HCV replication, yet many fundamental processes in the viral lifecycle remain uncharacterized. Using RNAi-based genetics and an infectious HCV cell culture system, we performed an unbiased genome-wide screen to identify host factors required for productive HCV infection. We applied a two-part screening protocol to identify host factors involved in the complete viral lifecycle, from viral entry to production of infectious virus. A validation screen was subsequently performed to minimize potential off-target effects. 512 genes were identified in the initial screen and 262 were confirmed by the validation assay. We identified 238 host susceptibility factors (HSFs) and 24 host resistance factors (HRFs), the majority of which were not previously linked to HCV. Of these 262 validated hits, 45 target late-stage viral infection. Integrative bioinformatics analyses of these host genes and other published database revealed a broad and complex dependency of HCV on cellular processes and molecular functions, and also implicated novel cellular signaling pathways modulating HCV infection. Several key pathways including TGF-beta, ErbB, MAPK, focal adhesion and ubiquitin proteolysis are particularly enriched in the bioinformatics analysis. By applying various virologic assays and molecular techniques, a comprehensive map of cellular pathways and machineries that are associated with each steps of HCV lifecycle, including viral entry, intracellular trafficking, viral RNA replication and translation, polyprotein processing, virion assembly and secretion, are being established. A global identification and characterization of HCV-host interactions will significantly advance our understanding of HCV-related pathogenesis, and hence illuminates potentially valuable targets for prophylactic and therapeutic interventions. One of the critical HCV host factors identified above is IKK-alpha. Here we describe a novel NF-KB-independent and kinase-mediated nuclear function of IKK-alpha in HCV assembly. HCV infection, through its viral pathogen-associated molecular pattern (PAMP), interacts specifically with DDX3 as a pattern recognition receptor (PRR) to activate IKK-alpha, which translocates to the nucleus and induces a CBP/p300-mediated transcriptional program involving SREBPs. This novel innate pathway induces lipogenic genes and enhances core-associated lipid droplet formation to facilitate viral assembly. Chemical inhibitors of IKK-alpha suppress HCV infection and IKK-alpha-induced lipogenesis, offering a proof-of-concept approach for novel HCV therapeutic development. Our results provide a conceptual advance in revealing that HCV commands a novel mechanism to its own advantage through exploiting the intrinsic innate immunity and hijacking the lipid metabolism, which likely contributes to a high rate of chronicity and the pathological hallmark of steatosis in HCV infection. Using the same screening technology, we performed an unbiased strategy to identify cellular miRNAs associated with HCV infection and functionally interrogate these miRNAs with our previous HCV small interference RNA (siRNA) screen database to derive an extensive cellular/viral regulatory network in productive HCV infection. We performed a combined genome-wide miRNA (1000 miRNA in miRBase Sequence 13.0) mimic-inhibitor screen by using a two-part immunostaining format. In the primary screen, we identified 100 miRNAs that either reduced (antiviral) or enhanced (proviral) HCV infection. 60 of them were validated by a secondary screen using a luciferase reporter virus. 24 miRNAs were proviral and 36 antiviral. miR122 was a confirmed proviral miRNA in the screen and one other miRNA, miR196, recently shown to play a role HCV replication, was also a confirmed hit. By using various HCV model systems, the majority of these novel miRNAs can be assigned to different stages of HCV life cycle entry, IRES-mediated translation, viral RNA replication, and assembly/release. In addition, our global miRNA expression analyses in both Huh7.5.1 cells and primary human hepatocytes revealed that many miRNAs are regulated by HCV infection and some of them are also validated hits of the above genome-wide functional screen, suggesting a complicated interaction between miRNA regulation and HCV infection. We further characterized two of the validated miRNAs for their effects on HCV propagation and demonstrated that these miRNAs target certain host factors identified in our siRNA screen, potentially explaining the functional effects of these miRNAs on HCV infection. A comprehensive investigation of cellular miRNAs modulating the complete HCV life cycle will yield critical insights into HCV pathogenesis and provide novel therapeutic targets.